PROJECT SUMMARY Medulloblastoma is the most common malignant brain tumor in children. Current treatment includes radiation and multiple chemotherapies which cause severe acute and significant long term side effects. Despite this aggressive treatment, a subset of patients called ?Group 3 MYC-amplified medulloblastoma? have a survival rate less than 25%. We and others have shown that MYC-amplified cancers change their energy metabolic requirements and become ?glutamine addicted? for their growth and survival. We have developed syngeneic human neural stem cell models which are +/- MYC. Recently we found that the MYC-amplified cells are exquisitely sensitive to the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON), while equally aggressive cells without MYC expression as well normal human neural stem cells are unaffected. While promising, DON is not clinically available. Its development was halted due to gastrointestinal (GI) toxicities, as the GI system is highly dependent on glutamine utilization. Moreover, DON's brain penetration is limited. To overcome DON's peripheral toxicities and to enhance its delivery to brain tumors, we created unique DON prodrugs. They were designed to circulate intact as inert prodrugs in plasma, but permeate and be cleaved to release DON once inside the brain. Compared to equimolar doses of DON, when our lead prodrug JHU-333 was evaluated in vivo (mice, swine, and primates) it resulted in significantly less DON exposure in plasma, and was preferentially biotransformed to DON in the brain, providing a 7-10-fold improvement in the brain/plasma ratio with substantially less GI toxicity. When tested in our MYC-amplified Group 3 orthotopic xenograft model, our brain-penetrable DON prodrug significantly increased survival following oral administration without overt toxicity. Although promising, JHU-333 is not ideal for translational as it exhibits high clearance with a short t1/2 (0.5-2hr). Thus, our main drug discovery focus will be to create metabolically stable DON prodrugs that permeate and are retained in the brain so that their target inhibition can be sustained. In this grant, two PIs with complimentary expertise will design novel DON prodrugs with optimized pharmacokinetic parameters and characterize their efficacy/toxicity profiles in Group 3 MYC-amplified medulloblastoma mouse models. At the completion of these studies we will have developed novel, robust, brian penetrant, and safe inhibitors of glutamine metabolism, laying the ground-work for their rapid introduction into clinical trials.